Neutrophils and macrophages are the primary cellular mediators of immediate host defense against bacterial nfection. However, uncontrolled activation of these cells contributes significantly to tissue injury in autoimmune and inflammatory disease. The Src-family and Syk tyrosine kinases are central regulators of neutrophil activation. In previous studies supported by this project, we found that these kinases play a critical role in leukocyte (31, (32 and (33 integrin signaling. Knockout mice lacking the myeloid expressed Src-family kinases (hck-/-fgr-/-lyn-/-) or Syk (syk-/-) fail to respond to integrin stimulation, leading to blocked respiratory burst, degranulation and cell spreading responses. Our preliminary studies of integrin "outside-in" signaling suggest that the kinases are operating in a pathway similar to classical immunoreceptor signaling, based on the observation that mice lacking the immunoreceptor adapters DAP 12 and FcRy have the same integrin signaling defects as the kinase knockouts. In aim#l of this proposal we will test the hypothesis that neutrophil "outside-in" integrin signaling utilizes an immunoreceptor pathway by retroviral gene transduction of mutant kinases or adapters into hematopoietic stem cells from knockout mice. We will use TAT-mediated protein transduction methods and expression of fluorescent activation reporters to study RhoGTPase activation in neutrophil integrin signaling. Because mutation of these kinases affects lymphocyte function, it has been difficult to study the specific role of Src-family or Syk kinases in innate immune response during inflammation or infection. In aim#2, we will use myeloid specific conditional knockout mice lacking Lyn or Syk kinase to study the roles of these enzymes in inflammation models and in host defense to infection, using an NIAID class B agent. Studies of knockout mice are complicated by the problem that single mutations often produce global changes in gene expression during leukocyte development. Thus one is often comparing cells of different developmental states. In aim#3, we will use a chemical-genetic approach to avoid this problem, by generating knock-in mice with point mutations in either Lyn or Syk that render the enzymes uniquely susceptible to highly specific chemical inhibitors. We can then use these inhibitors to acutely inactivate protein function in isolated cells or in vivo. Our studies will depend heavily on knockout/knock-in mouse models and use of primary cells thus ensuring physiological significance. These studies will better define the roles of Src-family and Syk kinases in innate immune function, contributing significantly to our mechanistic understanding of immune response in biodefense in emerging infectious disease.